Multi-drug resistance (MDR) in Gram-negative pathogens, including the Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter spp., and Stenotrophomonas maltiphilia, pose a significant threat to the effective treatment of infections caused by these organisms (Kibbey, et al.: An integrated process for measuring the physicochemical properties of drug candidates in a preclinical discovery environment. In J Pharm Sci, vol. 90, pp. 1164-1175, (2001); Kang et al.: Risk factors for antimicrobial resistance and influence of resistance on mortality in patients with bloodstream infection caused by Pseudomonas aeruginosa. In Microb Drug Resist, vol. 11, pp. 68-74, (2005); Kang, et al.: Clinical epidemiology of ciprofloxacin resistance and its relationship to broad-spectrum cephalosporin resistance in bloodstream infections caused by Enterobacter species. In Infect Control Hosp Epidemiol, vol. 26, pp. 88-92, (2005); Kang, et al.: Bloodstream infections caused by antibiotic resistant Gram-negative bacilli: risk factors for mortality and impact of inappropriate initial antimicrobial therapy on outcome Antimicrob Agents Chemother, vol. 49, pp. 760-766, (2005)). The MDR threat has been exacerbated by the recent decrease in commercial efforts to discover and develop new antibacterial agents. In addition, antibacterial agents that have been introduced recently into the clinic or are in development, such as daptomycin, gemifloxacin, telithromycin, and telavancin, are not active against Gram-negative pathogens. Recently approved agents with activity against Gram-negative bacteria include tigecycline and doripenem. While tigecycline is active against bacteria producing a tetracycline-specific pump in vitro, it is pumped out rapidly by the ubiquitous multidrug pumps, and its pharmacokinetic properties will limit its use for treating urinary tract and bloodstream infections (Peleg, et al.: Hospital acquired infections due to Gram-negative bacteria. In N Engl J Med, vol. 362, pp. 1804-1813, (2010)), as will the evolution of resistance during therapy (Anthony, et al.: Clinical and microbiological outcomes of serious infections with multidrug-resistant Gram-negative organisms treated with tigecycline. Iu Clin Infect Dis, vol. 46, pp. 567-570, (2008)). Clearly, novel strategies for effectively treating infections caused by MDR Gram-negative pathogens are urgently needed.
The MDR phenotype has been attributed to both acquired and intrinsic mechanisms of resistance. Acquired resistance mechanisms include mutations that decrease the affinity of the target for an antibacterial agent, or through acquisition of mobile genetic elements that modify the target or inactivate the antibacterial agent. In recent years, the importance of the role that intrinsic resistance mechanisms play in the development of the MDR phenotype has been fully appreciated (Nikaido, et al.: Broad-specificity efflux pumps and their role in multidrug resistance of Gram-negative bacteria. In FEMS Microbiol Rev, vol. 36, pp. 340-363, (2012); Poole: Bacterial stress responses as determinants of antimicrobial resistance. In J Antimicrob Chemother, vol. 67, pp. 2069-2089, (2012)). Recent genome-wide screens for mutants with altered susceptibilities to antibacterial agents have identified several genes that play a role in intrinsic resistance in E. coli (Tamae, et al.: Determination of antibiotic hypersensitivity among 4,000 single-gene-knockout mutants of Escherichia coli. In J Bacteriol, vol. 190, pp. 5981-5988, (2008)) and P. aeruginosa (Breidenstein, et al.: Complex ciprofloxacin resistome revealed by screening a Pseudomonas aeruginosa mutant library for altered susceptibility. In Antimicrob Agents Chemother, vol. 52, pp. 4486-4491, (2008); Fajardo, et al.: The neglected intrinsic resistome of bacterial pathogens. In PLoS One, vol. 3, pp. e1619, (2008); Gallagher, et al.: Genome-scale identification of resistance functions in Pseudomonas aeruginosa using Tn-seq. In MBio, vol. 2, pp. e00315-00310, (2012)), including genes involved in heat shock, SOS response, membrane stress response, and efflux. A network of proteases FtsH and accessory proteins YccA, HtpX, HflK, HslUV, and HflC are involved in intrinsic resistance to aminoglycoside tobramycin (Hinz, et al.: Membrane proteases and aminoglycoside antibiotic resistance. In J Bacteriol, vol. 193, pp. 4790-4797, (2011)), presumably by removing misfolded proteins. In addition, the SOS has been implicated in intrinsic resistance to quinolone antibiotics (Piddock, et al.: Bactericidal activities of five quinolones for Escherichia coli strains with mutations in genes encoding the SOS response or cell division. In Antimicrob Agents Chemother, vol. 36, pp. 819-825, (1992)). However, the RND efflux pumps of Gram-negative bacteria play a major role in MDR. Because of their broad substrate specificity, overexpression of these efflux pumps results in decreased susceptibility to a wide range of antibacterial agents and biocides (Nikaido, et al.: Broad-specificity efflux pumps and their role in multidrug resistance of Gram-negative bacteria. In FEMS Microbiol Rev, vol. 36, pp. 340-363, (2012)).
The major efflux pump of E. coli is a typical Resistance-Nodulation-Division (RND) pump, which is a tripartite structure consisting of an integral membrane efflux transporter with broad substrate specificity (AcrB), an outer membrane channel (TolC), and a periplasmic protein adapter (AcrA). Antibiotics enter the periplasmic space through a porin or by diffusion through the lipid bilayer, where they interact with the substrate binding pocket of AcrB. The AcrB transporter uses proton motive force to extrude the compound into the TolC channel and to the exterior. These RND family pumps not only produce intrinsic levels of resistance to antibacterial agents, including the fluoroquinolones (FQs; e.g. ciprofloxacin and levofloxacin) and β-lactams (e.g., piperacillin, meropenem, and aztreonam) (Piddock: Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria. In Clin Microbiol Rev, vol. 19, pp. 382-402, (2006)), but also produce an MDR phenotype when overproduced. In addition, inhibition of RND pumps in P. aeruginosa by genetic deletion (Lomovskaya, et al.: Use of a genetic approach to evaluate the consequences of inhibition of efflux pumps in Pseudomonas aeruginosa. In Antimicrob Agents Chemother, vol. 43, pp. 1340-1346, (1999)) or with a potent efflux pump inhibitor (EPI) (Lomovskaya, et al.: Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy. In Antimicrob Agents Chemother, vol. 45, pp. 105-116, (2001)) decreases the frequency of resistance to levofloxacin, and AcrAB-TolC is required for selection of target mutations for FQ resistance in E. coli (Singh, et al.: Temporal interplay between efflux pumps and target mutations in development of antibiotic resistance in Escherichia coli. Iu Antimicrob Agents Chemother, vol. 56, pp. 1680-1685, (2012)). In addition, RND pumps have been shown to play a role in virulence of the enteric pathogen Salmonella enterica serovar Typhimurium (Nishino, et al.: Virulence and drug resistance roles of multidrug efflux systems of Salmonella enterica serovar Typhimurium. In Mol Microbiol, vol. 59, pp. 126-141, (2006)), and EPIs that target RND pumps have been shown to inhibit biofilm formation in E. coli and K. pneumoniae (Kvist, et al.: Inactivation of efflux pumps abolishes bacterial biofilm formation. In Appl Environ Microbiol, vol. 74, pp. 7376-7382, (2008)). Therefore, EPIs could be used as adjunctive therapies with an FQ or β-lactam antibiotic to improve antibacterial potency at low antibiotic concentrations, prevent the emergence of resistance, inhibit biofilm formation, and decrease virulence of enteric pathogens.
Several potent efflux pump inhibitors have been described in the literature (Thorarensen, et al.: 3-Arylpiperidines as potentiators of existing antibacterial agents. In Bioorg Med Chem Lett, vol. 11, pp. 1903-1906, (2001); Lomovskaya, et al.: Practical applications and feasibility of efflux pump inhibitors in the clinic—a vision for applied use. In Biochem Pharmacol, vol. 71, pp. 910-918, (2006); Mahamoud, et al.: Quinoline derivatives as promising inhibitors of antibiotic efflux pump in multidrug resistant Enterobacter aerogenes isolates. In Curr Drug Targets, vol. 7, pp. 843-847, (2006); Mahamoud, et al.: Antibiotic efflux pumps in Gram-negative bacteria: the inhibitor response strategy. In J Antimicrob Chemother, vol. 59, pp. 1223-1229, (2007)), however, none have reached clinical development. A family of peptidomimetics, including PAβN (MC-207 110), that exhibited potent inhibition of efflux pumps in P. aeruginosa has been developed for use as an adjunctive therapy (Renau, et al.: Inhibitors of efflux pumps in Pseudomonas aeruginosa potentiate the activity of the fluoroquinolone antibacterial levofloxacin. In J Med Chem, vol. 42, pp. 4928-4931, (1999); Lomovskaya, et al.: Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy. In Antimicrob Agents Chemother, vol. 45, pp. 105-116, (2001); Renau, et al.: Addressing the stability of C-capped dipeptide efflux pump inhibitors that potentiate the activity of levofloxacin in Pseudomonas aeruginosa. In Bioorg Med Chem Lett, vol. 11, pp. 663-667, (2001); Renau, et al.: Peptidomimetics of efflux pump inhibitors potentiate the activity of levofloxacin in Pseudomonas aeruginosa. In Bioorg Med Chem Lett, vol. 12, pp. 763-766, (2002); Renau, et al.: Conformationally restricted analogues of efflux pump inhibitors that potentiate the activity of levofloxacin in Pseudomonas aeruginosa. In Bioorg Med Chem Lett, vol. 13, pp. 2755-2758, (2003); Watkins, et al.: The relationship between physicochemical properties, in vitro activity and pharmacokinetic profiles of analogues of diamine-containing efflux pump inhibitors. In Bioorg Med Chem Lett, vol. 13, pp. 4241-4244, (2003); Yoshida, et al.: MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 5: Carbon-substituted analogues at the C-2 position. In Bioorg Med Chem, vol. 14, pp. 1993-2004, (2006); Yoshida, et al.: MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 6: exploration of aromatic substituents. In Bioorg Med Chem, vol. 14, pp. 8506-8518, (2006); Yoshida, et al.: MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 7: highly soluble and in vivo active quaternary ammonium analogue D13-9001, a potential preclinical candidate. In Bioorg Med Chem, vol. 15, pp. 7087-7097, (2007)). In addition, pyridopyrimidine EPIs that are specific for the MexAB efflux pump of P. aeruginosa were advanced to the preclinical stage (Nakayama, et al.: MexAB-OprM-specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 1: discovery and early strategies for lead optimization. In Bioorg Med Chem Lett, vol. 13, pp. 4201-4204, (2003); Nakayama, et al.: MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 2: achieving activity in vivo through the use of alternative scaffolds. In Bioorg Med Chem Lett, vol. 13, pp. 4205-4208, (2003); Nakayama, et al.: MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 3: Optimization of potency in the pyridopyrimidine series through the application of a pharmacophore model. In Bioorg Med Chem Lett, vol. 14, pp. 475-479, (2004); Nakayama, et al.: MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 4: Addressing the problem of poor stability due to photoisomerization of an acrylic acid moiety. In Bioorg Med Chem Lett, vol. 14, pp. 2493-2497, (2004); Yoshida, et al.: MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 5: Carbon-substituted analogues at the C-2 position. In Bioorg Med Chem, vol. 14, pp. 1993-2004, (2006); Yoshida, et al.: MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 6: exploration of aromatic substituents. In Bioorg Med Chem, vol. 14, pp. 8506-8518, (2006); Yoshida, et al.: MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 7: highly soluble and in vivo active quaternary ammonium analogue D13-9001, a potential preclinical candidate. In Bioorg Med Chem, vol. 15, pp. 7087-7097, (2007)). Some of these inhibitors were validated using in vivo infection models (Nakayama, et al.: MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 2: achieving activity in vivo through the use of alternative scaffolds. In Bioorg Med Chem Lett, vol. 13, pp. 4205-4208, (2003); Yoshida, et al.: MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 7: highly soluble and in vivo active quaternary ammonium analogue D13-9001, a potential preclinical candidate. In Bioorg Med Chem, vol. 15, pp. 7087-7097, (2007)); however, they were abandoned because of toxicity problems (Lomovskaya, et al.: Practical applications and feasibility of efflux pump inhibitors in the clinic—a vision for applied use. In Biochem Pharmacol, vol. 71, pp. 910-918, (2006)).
The following table lists the classes of reported EPIs and references.
Compound ClassReferences1PeptidomimeticsRenau, et al., J Med Chem, vol. 42, pp. 4928-4931, (1999);Lomovskaya, et al., Antimicrob Agents Chemother, vol. 45, pp. 105-116, (2001);Renau, et al., Bioorg Med Chem Lett, vol. 11, pp. 663-667, (2001);Renau, et al., Bioorg Med Chem Lett, vol. 12, pp. 763-766, (2002);Renau, et al., Bioorg Med Chem Lett, vol. 13, pp. 2755-2758, (2003);Watkins, et al., Bioorg Med Chem Lett, vol. 13, pp. 4241-4244, (2003);Yoshida, et al., Bioorg Med Chem, vol. 14, pp. 1993-2004, (2006);Yoshida, et al., Bioorg Med Chem, vol. 14, pp. 8506-8518, (2006);Yoshida, et al., Bioorg Med Chem, vol. 15, pp. 7087-7097, (2007))2PyridopyrimidinesNakayama, et al., Bioorg Med Chem Lett, vol. 13, pp. 4201-4204,(2003);Nakayama, et al., Bioorg Med Chem Lett, vol. 13, pp. 4205-4208,(2003);Nakayama, et al., Bioorg Med Chem Lett, vol. 14, pp. 475-479, (2004);Nakayama, et al., Bioorg Med Chem Lett, vol. 14, pp. 2493-2497,(2004);Yoshida, et al., Bioorg Med Chem, vol. 14, pp. 1993-2004, (2006);Yoshida, et al., Bioorg Med Chem, vol. 14, pp. 8506-8518, (2006);Yoshida, et al., Bioorg Med Chem, vol. 15, pp. 7087-7097, (2007)3QuinolinesChevalier, et al., J Med Chem, vol. 44, pp. 4023-4026, (2001);Mallea, et al., Biochem J, vol. 376, pp. 801-805, (2003);Chevalier, et al., Antimicrob Agents Chemother, vol. 48, pp. 1043-1046,(2004);Mahamoud, et al., Curr Drug Targets, vol. 7, pp. 843-847, (2006)4QuinazolinesChevalier, et al., J Antimicrob Agents, vol. 36, pp. 164-168, (2010);Mahamoud, et al., Microbiology, vol. 157, pp. 566-571, (2011))53-ArylpiperidinesThorarensen, et al., In Bioorg Med Chem Lett, vol. 11, pp. 1903-1906,(2001);Bohnert, et al., Antimicrob Agents Chemother, vol. 49, pp. 849-852,(2005)6Repurposed drugs Piddock, et al., J Antimicrob Chemother, vol. 65, pp. 1215-1223, (2010);Bohnert, et al., J Antimicrob Chemother, vol. 66, pp. 2057-2060, (2011);Li, et al., J Antimicrob Chemother, vol. 66, pp. 769-777, (2011)
The need remains for more effective efflux pump inhibitors as a means of combatting bacterial infection.